Method and system for rapid parasite detection

ABSTRACT

The invention described herein relates to a method of detecting malaria comprising the steps of: (i) delivering an evanescent IR beam through said ATR substrate in contact with a patient blood sample; (ii) detecting IR radiation transmitted from the ATR substrate to produce a signal characteristic for one or more lipids in the sample, and (iii) processing said signal and a set of reference library spectra of lipids associated with malaria parasites in order to detect matches and quantify said one or more lipids in the sample. In contrast to the prior art, the present invention relies on detecting lipids instead of hemozoin.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is continuation of U.S. application Ser. No.15/117,149, filed on Aug. 5, 2016, now U.S. Pat. No. 9,983,130, which isa U.S. National Phase of International Patent Application Serial No.PCT/AU2014/000080, entitled “METHOD AND SYSTEM FOR RAPID MALARIADETECTION”, filed on Feb. 5, 2014, the entire contents of which arehereby incorporated by reference in their entirety for all purposes.

FIELD OF USE

The present invention relates to the field of malaria detection,particularly detection and quantification of early stage malariaparasites in infected cells.

In one form, the invention relates to a method of using Attenuated TotalReflection Infrared (ATR-IR) spectroscopy for detection andquantification of malaria.

In another form, the invention relates to a method of multivariateanalysis for analysis of data obtained by ATR-IR.

In one particular aspect the present invention is suitable for use fordiagnosis of malarial infection.

It will be convenient to hereinafter describe the invention in relationto field use of the present invention, however it should be appreciatedthat the present invention is not limited to that use only and thepresent invention can be adapted for use in a range of locationsincluding laboratories, and in a range of sizes from bench scale highthroughput diagnostic machines of the type used in commercial pathologylaboratories.

BACKGROUND

Throughout this specification the use of the word “inventor” in singularform may be taken as reference to one (singular) inventor or more thanone (plural) inventor of the present invention.

It is to be appreciated that any discussion of documents, devices, actsor knowledge in this specification is included to explain the context ofthe present invention. Further, the discussion throughout thisspecification comes about due to the realisation of the inventor and/orthe identification of certain related art problems by the inventor.Moreover, any discussion of material such as documents, devices, acts orknowledge in this specification is included to explain the context ofthe invention in terms of the inventor's knowledge and experience and,accordingly, any such discussion should not be taken as an admissionthat any of the material forms part of the prior art base or the commongeneral knowledge in the relevant art in Australia, or elsewhere, on orbefore the priority date of the disclosure and claims herein.

Attenuated Total Reflection Infrared (ATR-IR) Spectroscopy

Spectroscopy is the branch of science devoted to discovering thechemical composition of materials by examining the interaction ofelectromagnetic radiation with the material. Infrared (IR) spectroscopyrelates primarily to the absorption of energy by molecular vibrationshaving wavelengths in the infrared segment of the electromagneticspectrum, that is energy of wave number between 200 and 4000 cm⁻¹. Ramanspectroscopy relates to the inelastic scattering of monochromatic lightgiving wavelength shifts that depend on the molecular vibrations, havingtypically wave number shifts between 20 and 4000 cm⁻¹.

ATR is a sampling technique that can be used in conjunction with IR. ATRspectroscopy offers the advantages of being potentially portable, it isinexpensive and thus has become a very powerful tool in the analysis ofbiological cells and tissues. ATR also allows samples to be examineddirectly in the solid or liquid state without further preparation, andcompared with transmission-IR, the path length into the sample isshorter, avoiding strong attenuation of the IR signal in highlyabsorbing media such as aqueous solutions.

In use, the sample is put in contact with the surface of a crystalhaving a higher refractive index than the sample. A beam of IR light ispassed through the ATR crystal in such a way that it reflects at leastonce off the internal surface in contact with the sample. Thisreflection forms an evanescent wave which extends into the sample. Thepenetration depth into the sample depends on the wavelength of light,the angle of incidence and the indices of refraction for the ATR crystaland the medium being probed. The number of reflections may be varied.The beam is then collected by a detector as it exits the crystal.

Malaria

Malaria is a mosquito borne disease caused by parasitic protozoans ofthe genus Plasmodium. Five species of Plasmodium can infect humans—P.falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi—but the vastmajority of deaths are caused by P. falciparum. P. falciparum causes upto 1.2 million fatalities per annum. Accurate and early diagnosisfollowed by the immediate treatment of the infection is essential toreduce mortality and prevent overuse of antimalarial drugs.

New technologies to diagnose malaria must be cost effective and havehigh sensitivity and be able to detect circulating stages of the malariaparasite namely the ring and gametocyte forms because these are the onlystages present in peripheral blood circulation.

The current suite of malarial diagnostics in clinical use include: (i)optical microscopy of thick blood films, (ii) Rapid Diagnostic Tests(RDTs) based on the detection of antigens specific to P. falciparum,(iii) gene amplification techniques such as polymerase chain reaction(PCR) and (iv) serological detection tests using antibodies such asimmunofluorescence (IFA) and enzyme-linked immunosorbent assay (ELISA).

Each method has its own advantages and disadvantages. For example,optical microscopy requires preparation of blood smear samples usingreagents and is based on visual assessment of the morphology of bloodcells. The method is inherently subjective and requires experiencedmicroscopists to make diagnosis.

Polymerase chain reaction (PCR) is considered the most sensitive andspecific method, but has the drawbacks of being time consuming,technically sophisticated, expensive, and requiring a PCR machine, andis thus not suitable for malaria diagnosis in remote areas. MalariaRDTs, which are based on capture of parasite antigens by monoclonalantibodies incorporated into a test strip, are easy to use but areunable to quantify parasitemia.

A review of existing methods indicates that the examination of stainedblood smears by light microscopy remains the method of choice formalaria diagnosis because it is inexpensive and has good sensitivity(5-10 parasites/μl blood). However, it is labor-intensive, lengthy, andmore importantly, requires skilled and experienced microscopists, and isincreasingly burdensome as malaria rates decline with most smearsexamined being negative.

During the course of its life the malaria parasite transgresses throughseveral developmental stages including a sexual and an asexualreproductive pathway. The sexual or progeny phase, which occurs withinthe gut of female Anopheles mosquito, produces numerous infectious formsknown as sporozoites that are transferred to the mosquito salivaryglands and injected into the human host during a blood meal.

Sporozoites that enter a blood vessel move to the liver and invadehepatocytes where they develop into schizonts each containing tens ofthousands of merozoites. The merozoites are subsequently released andinvade the erythrocytes initiating the intraerythrocytic asexual phaseof the life cycle. The merozoites grow and divide in the food vacuoleand progress through three distinct morphological phases known as thering, trophozoite and schizont stages (FIG. 1).

Mature stage parasites adhere to the vascular endothelium and thus onlyring stage parasites are observed in blood smears. The schizonts burst,releasing the merozoites and the intraerythocytic cycle continues.Instead of replicating, some merozoites in the erythrocytes develop intosexual forms of the parasite, called male and female gametocytes, thatare capable of undergoing transmission to mosquitoes.

Early stage gametocytes sequester away from the peripheral circulationbut late stage gametocytes are present in blood smears, and gametocytecarriage underpins endemicity of disease. The detection of the rings inperipheral blood is critical for early diagnosis and treatment. Thedetection of low levels of gametocytes in asymptomatic long-term malariacarriers is critical to efforts to eradicate malaria.

During the intraerythrocytic stages of the parasites life cycle P.falciparum endocytoses packets of host cell cytoplasm, catabolizes thelipids and hemoglobin and in the process releases free heme, which istoxic to the organism. The malaria parasite has evolved a detoxificationpathway that uses the lipid by-products to catalyze the sequestration offree heme into an insoluble pigment known as hemozoin (Hz). Hence Hz isa disposal product formed from the digestion of blood by malariaparasites (and some other blood feeding parasites).

Synchrotron powder diffraction analyses have shown that crystals of Hz(and its synthetic equivalent β-hematin) are composed of a repeatingarray of iron-carboxylate interacting heme dimers, stabilized byhydrogen bonding and π-π interactions.

Vibrational spectroscopic techniques have been used extensively inunderstanding the molecular and electronic structure of β-hematin andHz; however, the use of vibrational spectroscopy for malaria diagnosticshas not been fully exploited. Raman imaging microscopy has been exploredas a potential non-subjective method to diagnose malaria parasites basedon the strong scattering from the Hz pigment. (Wood et al, ResonanceRaman microscopy in combination with partial dark-field microscopylights up a new path in malaria diagnostics, Analyst 2009, 134.1119-1125). While the technique has shown potential to detect ring formsof the parasite the time taken to record an image is on the order ofseveral hours and therefore not suitable for the clinical environment.

Efforts have also been made to investigate the potential of synchrotronFourier Transform Infrared (FTIR) in combination with PrincipalComponent Analysis (PCA) to differentiate between intraerythrocyticstages of the parasite life cycle based on the molecular signatures ofHz and specific lipids (Webster et al. Discriminating theIntraerythrocytic Lifecycle Stages of the Malaria Parasite UsingSynchrotron FT-IR Microspectroscopy and an Artificial Neural Network.Analytical Chemistry 2009, 81. 2516-2524). Webster et al found that asthe parasite matures from its early ring stage to the trophozoite andfinally to the schizont stage there is an increase in absorbance andshifting of specific lipid bands.

This work demonstrated the potential of using FTIR spectroscopy as adiagnostic tool for malaria but clearly a synchrotron-based method isnot suitable for routine laboratory use.

In particular, malaria detection methods of the prior art have focussedon detection of Hz. However, one of the principal problems with relyingsolely on the detection of Hz is that early forms of the malariaparasite (the ring stage) have very small amounts of Hz. Thus, manyRaman methods of the prior art can optimally detect trophozoites whichhave large amounts of Hz, however this suffers the drawback thattrophozoites are not generally found in peripheral blood.

SUMMARY OF INVENTION

An object of the present invention is to provide a method of malariadetection and quantification suitable for laboratory or field use.

A further object of the present invention is to alleviate at least onedisadvantage associated with the related art.

It is an object of the embodiments described herein to overcome oralleviate at least one of the above noted drawbacks of related artsystems or to at least provide a useful alternative to related artsystems.

In a first aspect of embodiments described herein there is provided amethod of detecting malaria comprising the steps of:

-   -   (i) delivering an evanescent IR beam through said ATR substrate        in contact with a patient blood sample;    -   (ii) detecting IR radiation transmitted from the ATR substrate        and producing one or more signals characteristic of one or more        lipids in the sample, and    -   (iii) processing said one or more signals to identify said one        or more lipids and any malaria parasite with which they are        associated.

Preferably the processing step includes comparing the one or moresignals with a set of reference library spectra of lipids associatedwith malaria parasites in order to detect matches and quantify said oneor more lipids in the sample. The reference library may include a widerange of spectral information including characteristic lipid profilesfor lipids associated with each stage of the malaria parasite's lifecycle, and control samples of infected and uninfected RBCs. Furthermore,given inherent differences in their characteristics, single cellprofiles as well as profiles for broad populations of cells arepreferably included in the library.

In contrast to methods of the prior art, the present invention does notrely on detecting Hz. Instead it is focussed on the characteristicspectroscopic lipid signatures expressed by malaria parasites atdifferent stages of their life cycle. The lipid signature detected byATR-IR is matched with a known spectroscopic lipid signature in alibrary.

Typically, the patient sample is derived from a small sample of bloodremoved by pin prick or syringe or the like. The blood may be applieddirectly to the ATR substrate, but more preferably, the red blood cellsare at least partially concentrated by separation from the rest of theblood sample.

The ATR substrate may be of any suitable type known in the technology,but typical substrates include crystals of germanium, zinc selenide ordiamond. In a preferred embodiment the ATR substrate for use in thecurrent method is diamond.

The lipids are typically associated with at least one or more of thefollowing: P. falciparum, P. vivax, P. ovale, P. malariae and P.knowlesi.

The method according to the present invention is relatively sensitive.It is capable of detecting a parasite level of 0.001% (better than 50parasites/μl blood; p-value=0.0006) or more in a volume of 1 mL blood.More preferably it is capable of detecting a parasite level of at least100 parasites/μl of sample.

Typically the IR spectroscopy is FTIR, that is, the raw data has beenconverted into a spectrum by the mathematical process called the Fouriertransform. FTIR spectrometers simultaneously collect spectral data in awide spectral range. The alternative, known as dispersive spectrometrymeasures intensity over a narrow range of wavelengths at a time but isvirtually obsolete.

The processing step of the present invention may be carried out byvarious techniques. In a preferred embodiment, the processing comprisesconverting the ATR-IR spectrum into a second derivative, then applying apartial least squares regression model generated by using a librarycomprising a calibration set of spectral standards containing mixturesof normal and infected RBC at different ratios.

Any suitable algorithm known in the art may be used to convert thespectrum into a second derivative. This removes baseline offsets andresolve inflection points in the spectral bands. Preferably, the secondderivative spectrum is then run through a partial least squares (PLS)regression model generated by using a calibration set of spectralstandards containing mixtures of normal and infected RBC at differentratios. However many suitable algorithms other than PLS will be readilyapparent to the person skilled in the art.

In a second aspect of embodiments described herein there is provided asystem of analysis for diagnosing malaria, the system comprising:

-   -   an ATR substrate for receiving a blood sample,    -   an FTIR spectrometer for delivering an evanescent IR beam        through said ATR substrate,    -   a detector for detecting IR radiation transmitted from the ATR        substrate to produce a signal,    -   a processor for processing said signal to create an FTIR        spectrum,        wherein in use an FTIR spectrum of a blood sample is compared        with a library of known spectroscopic signatures of lipids        associated with malaria parasites to identify matches.

The method and system of the present invention may thus be associatedwith a software platform that submits the results of ATR-IR tocomparison with a library of ATR-IR results for strains of malariaparasites at different stages of development. The library may be localor remote. For example, a spectrum recorded according to the presentinvention may be submitted from a remote location to a server foridentification of matches between the spectrum and a library of knownspectroscopic signatures of lipids associated with any malaria parasitesin the RBC.

The method or system of the present invention may be used in combinationwith multivariate statistics or neural network methods to identify themalarial strain associated with the spectroscopic signature of a lipidin the blood sample. Neural networks are computational models that arecapable of machine learning and pattern recognition and are particularlywell adapted for classification, including pattern and sequencerecognition and fitness approximation.

In another aspect of embodiments described herein there is provided aprocessor means adapted to operate in accordance with a predeterminedinstruction set, said apparatus, in conjunction with said instructionset, being adapted to perform the method according to the presentinvention.

Other aspects and preferred forms are disclosed in the specificationand/or defined in the appended claims, forming a part of the descriptionof the invention.

In essence, embodiments of the present invention stem from therealization that parasite-specific lipid profiles could be used todetect and identify specific strains of malarial infection. This is asignificant deviation from the wisdom of the prior art which focuses onthe parasite-specific nature of hemozoin production, particularlydetection of hematin (monomeric precursor) or hemozoin.

Advantages provided by the present invention comprise the following:

-   -   detection and quantification of malaria parasites can be rapidly        carried out,    -   can detect the early stages of the malaria parasite life cycle;    -   does not rely on detecting Hz and be used to detect forms of the        malaria parasite such as ring and gametocytes that have small,        or negligible quantities of Hz;    -   no cell counting or chemical treatment is required;    -   sample preparation time is minimal (<3 mins per sample);    -   the method is simple and inexpensive;    -   high sensitivity—a parasite level of 0.001% (better than 50        parasites/μl blood; p-value=0.0006) in a volume of 1 mL blood is        required;    -   the amount of patient blood required is minimal (approx 10 μl)—a        suitable amount can be obtained by pin-prick (which takes        approx. 25 μl);    -   avoids bias and errors associated with human interpretation.

Further scope of applicability of embodiments of the present inventionwill become apparent from the detailed description given hereinafter.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the disclosure hereinwill become apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee.

Further disclosure, objects, advantages and aspects of preferred andother embodiments of the present application may be better understood bythose skilled in the relevant art by reference to the followingdescription of embodiments taken in conjunction with the accompanyingdrawings, which are given by way of illustration only, and thus are notlimitative of the disclosure herein, and in which:

FIG. 1 is a diagram illustrating the asexual and sexual phases of themalaria parasite in the RBC. Merozoites (1) invade RBCs and developthrough the ring (3), trophozoite (5) (growing) and schizont (7)(dividing) stages. Some parasites differentiate to form male (9) andfemale (11) gametocytes that are capable of transmission to mosquitoes.Digestion of haemoglobin leads to the accumulation of Hz. Only ringstage parasites (3) and late gametocytes (9, 11) are present in theblood circulation.

FIGS. 2A and 2B illustrate ATR-FTIR average 2nd-derivative spectra forinfected RBCs (Ring, (3) Trophozoite (5), and Gametocyte (9) stages ofparasite) and uninfected RBCs (13) (control) of the C—H stretchingregion (FIG. 2A) and the Hz band marker range (FIG. 2B).

FIGS. 3A and 3B illustrate a PCA Scores Plot along PC1 and PC2 ofControl (C), Rings (R), Trophozoite (T) and Gametocytes (G) affected RBCdata sets (FIG. 3A) and a PC1 correlation Loadings Plot after a secondderivative function was applied to the C—H stretching region (3100-2800cm−1) (FIG. 3B).

FIGS. 4A and 4B illustrate a PCA Scores Plot (FIG. 4A) and the PC1correlation Loadings Plot (FIG. 4B) along PC1 and PC2 of Control—0% (13)& Rings 0.00001% (3), after a second derivative function was applied tothe C—H stretching region (3100-2800 cm−1).

FIG. 5 illustrates spectra of overlaid 2nd derivative spectra showingthe type of data used in the generating the calibration models. [Control(21), 0.05% (23), 0.8% (25), 0.5% (27), 3% (29)].

FIGS. 6A, 6B, 6C, 6D, 6E, and 6F illustrate regression plots for thecalibration and validation sets for three ranges of early ring stageparasitemia namely model 1 (FIG. 6A: 0, 10, 15, 20 and 30%), model 2(FIG. 6B: 0, 1, 1.75, 2.5, 3 and 5%) and model 3 (FIG. 6C: 0, 0.00001,0.005, 0.01, 0.05, 0.1, 0.2, 0.4, 0.5, 0.8 and 1%) along with thecorresponding regression co-efficient plots (FIGS. 6D, 6E and 6Frespectively) for 8-factors.

FIG. 7 illustrates example of predictions for unknown samples using PLSmodels in the range of 5-10% with the average standard error ofprediction of 0.08. The predicted values are shown as horizontal linesand the boxes around predicted value indicate the deviation.

FIG. 8 illustrates a system according to the present invention forATR-FTIR. The system includes a loading jig (31) for pipette (33) basedloading of about 10 μl of RBCs, separated from a whole blood sample bycentrifugation in an Eppendorf tube (35). The RBCs are loaded onto anATR substrate in the form of a diamond crystal (37). An evanescent IRbeam (39) is generated by an FTIR spectrometer and imposed on thediamond crystal (27) loaded with the RBC sample. A detector (41) detectsthe IR radiation transmitted from the diamond crystal (37) to produce asignal characteristic of one or more lipids present in the RBC sample.The resultant signal is passed on to a processor (43) for comparisonwith the library of malaria parasite associated lipids for diagnosis andfurther diagnosis.

DETAILED DESCRIPTION

The present invention will be illustrated with reference to theexperimental methods described below.

A system suitable for performing the method of the present invention wascreated by combining a standard bench top FTIR spectrometer and adiamond crystal ATR accessory as depicted in FIG. 8.

The ATR technique utilizes the property of total internal reflection togenerate an evanescent wave, which penetrates 2 to 3 μm into a sampleplaced in contact with the crystal face, depending on the wavelength,the refractive indices of the crystal and the sample, and the angle ofincidence of the infrared beam.

Typically, a suitable small sample of blood can be removed from apatient by pin prick or syringe or any other convenient method. Theblood may be applied directly to the ATR substrate, but more preferably,the red blood cells are at least partially concentrated by separationfrom the rest of the blood sample. This can be done by any convenientmeans such as centrifugation. The centrifugation step could be carriedout in the field using battery driven micro-centrifuges in an Eppendorftube.

An aliquot of packed RBCs in methanol is placed on the diamond window ofthe ATR accessory and rapidly dried with a blow dryer (1 minute). Whileother solvents are also suitable, methanol is particularly preferredbecause it facilitates the high sensitivity achieved with the ATR-FTIRapproach (see below).

The whole process of sample deposition and spectral recording is rapid,and can take less than 3 minutes using a single ATR element. Analgorithm converts the spectrum into a second derivative to removebaseline offsets and resolve inflection points in the spectral bands.The second derivative spectrum is then run through a partial leastsquares regression model generated by using a calibration set ofspectral standards containing mixtures of normal and infected RBC atdifferent ratios.

Plasmodium Culture and Gametocyte Enrichment

Plasmodium falciparum parasites (3D7 strain) were maintained aspreviously described (Foley et al, Photoaffinity labeling ofchloroquine-binding proteins in Plasmodium falciparum. J Biol Chem 1994,269. 6955-61). Briefly, parasites were maintained in O type human RBCs(sourced from the Australian Red Cross Blood Bank) and cultured inRPMI-HEPES medium supplemented with 5% human serum and 0.25% Albumax.Parasites were synchronized to ring stages by sorbitol lysis (C.Lambros, J. P. Vanderberg, Synchronization of Plasmodium falciparumerythrocytic stages in culture. J Parasitol 1979, 65. 418-20.) Highparasitemia ring stage cultures were obtained by seeding uninfected RBCswith purified schizont stage parasites and allowed to reinvade undershaking conditions overnight, reducing multiple infections. Parasitemiaswere calculated by Giemsa stained thin blood films, a minimum of 10fields of view were counted for each culture.

Accurate cell counts were obtained for uninfected and parasite infectedRBCs through counting on a hemocytometer. The dilutions were calculatedand samples prepared by diluting parasite-infected cultures withuninfected RBCs to obtain the desired dilution. All dilutions wereperformed in complete culture media, samples were then washed once in1×PBS, prior to fixation with cold methanol (EMPARTA ACS, Merck) on ice(<0° C.) and mixed thoroughly by pipetting. Samples were stored at 4° C.until analyzed.

Parasitemia Series

A series of infected methanol-fixed RBCs with cultured parasites atdifferent stages including rings, trophozoites, gametocytes at a rangeof parasitemia percentages (Table 1) were used to establish the PLScalibration models. Uninfected methanol-fixed RBCs were used as thecontrol (0% parasitemia).

TABLE 1 Parasitemia percentages of P. falciparum-infected RBCs atdifferent stages. Intra- Erythrocytic Stages Parasitemia series (%)Rings (a) 0.5, 1, 2.5, 5, 10, 15, 20, 30 Rings (b) 0.01, 1.75, 0.1, .08,0.2, 0.43, 7 Rings (2 series) (c) 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,3 Rings (2 series) (d) 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.01,0.1, 0.5, 1 Trophozoites 0.5, 1, 2.5, 5, 10, 20, 40, 50, 60, 80Gametocyte 0.09, 0.3, 0.18, 0.3, 0.75, 1.25, 2.5, 5, 10, 20, 40, 80

Equipment & Spectral Data Acquisition (ATR-FTIR Measurements)

A Bruker model EQUINOX 55-(Bruker Optic, Ettingen, Germany) FTIRspectrometer fitted with a N₂-cooled mercury-cadmium-tellurium (MCT)detector and a golden gate diamond ATR accessory (Specac limited,Orpington, Kent, UK) was used for spectral acquisition. The Brukersystem was controlled with an IBM-compatible PC running OPUS version 6.0software.

For each sample spectra 200 μL of the packed fixed cells were placed onthe diamond cell and air-dried with a blow dryer. Spectra were collectedwith a spectral resolution of 8 cm⁻¹ and 32 co-added interferogramsratioed against a clean diamond background. For each sample deposit, 3-5replicate spectra were recorded to assess precision and ensure thereproducibility of each sample spectrum.

Data Pre-Processing.

Pre-processing of the spectral data was performed in OPUS-(Bruker Optic,Ettingen, Germany) and the Unscrambler X (Version 10.0.1, Camo, Norway)software packages. For optimal modeling, raw spectra were vectornormalized and the second derivative calculated using the Savitzky-Golayalgorithm with 9 smoothing points.

Results and Discussion Fixative Selection Study

The following describes a preliminary study that was carried out toconfirm the application of the method of the present invention. The aimwas to optimize fixative types and explore spectral variations duringstorage times. Ethanol, methanol and formaldehyde fixatives wereexamined in the study. It was found that methanol is a preferredfixative for the ATR measurement according to the present invention asit gives more robust spectra (i.e. less variation during the storagetime) and the cells are easily separated from fixative withoutcentrifuging. Another advantage of methanol is that it evaporatesrapidly under a blow dryer and leaves no chemical residues. Air-dryingor fixing cells with glutaraldehyde does not achieve the samesensitivity and accurate quantification. The methanol may also assist inforcing dissolved lipids to the surface of the ATR crystal especiallywhen under pressure from the sample-clamping device.

It has been calculated that the ATR method according to the presentinvention can be used to detect lipid residues and Hz deposits from asfew as approximately 100 parasites on the ATR diamond crystal face at0.00001% parasitemia.

Spectral Precision/Reproducibility

Replicate spectra (30 in total from 6 sample deposits×5 spectra perdeposit) were obtained from all RBC samples to ensure thatrepresentative ATR-FTIR spectra were collected after the sample wasair-dried.

After pre-processing (normalization and derivative calculation)statistical tests were performed over the range of replicate spectra(600-4000 cm⁻¹) using Unscrambler X software. The descriptive statisticsdata including variance and standard deviations were used to assess thereproducibility of the IR spectra. As an example the replicate spectra(30 replicates) of the control sample showed a mean absorbance varianceof 0.0005. This confirmed the applicability of the method used fordeposition and drying of the sample deposit on the diamond crystal faceand indicated that the spectra of the dried sample deposits are robustand reproducible.

Overlaid Average Spectra

Replicate second derivative spectra from each stage of the parasiteslife cycle at different parasitemia percentages were averaged (usingreduced-average option in Unscrambler-X software) and overlaid (FIG. 2).

Replicate ATR-FTIR spectra from different stages of parasite infectedRBCs with the highest available parasitemia percentages [i.e. ring(30%), trophozoite (80%), gametocyte (40%) as well as control (0%)samples] were obtained. FIG. 2 shows the averaged second-derivativeoverlaid spectra of the C—H stretching region (3100-2800 cm⁻¹) as wellas the 1800-900 cm⁻¹ region highlighting the important Hz marker bandsfor infected RBCs from different parasite stages.

In the second derivate spectra the absorbance maxima become minima,therefore, in FIG. 2 the positive intensities for absorbance spectrabecome negative for second-derivative spectra. The CH stretching region(3100-2800 cm−1) is optimally diagnostic for different stages of theparasite as previously shown with synchrotron FTIR spectroscopy (Websteret al, Analytical chemistry 2009, 81. 2516-2524.)

There is also evidence for contributions from nucleic acids as evidencedby the phosphodiester marker bands including the asymmetric stretch at1241 cm⁻¹ and the symmetric stretch at 1095 cm⁻¹. The C—O stretchingvibration from the propionate group from Hz expected around 1208-1215cm⁻¹ is observed as a shoulder feature in the second derivative spectraof trophozoites and to a lesser extent in the gametocytes. In terms ofdiagnostic capability use of the CH stretching region was found toachieve a higher sensitivity compared to the 1800-950 cm⁻¹ region and acombination of both regions.

Principal Component Analysis (PCA)

PCA was performed on all the replicate RBC samples from individualstages of the parasite life cycle following spectral datapre-processing. PCA is one of the most powerful exploratory tools forlarge data set analysis. PCA reduces the dimensionality of the data setby decomposing the data set into a signal and noise part by findinglinear combinations of the original variables. PCA was applied tosecond-derivative ATR-FTIR spectra from infected RBCs including the ringtrophozoite and gametocyte stages as well as the control samples(uninfected RBCs) with the aim of assessing spectral variance across asub-population of cells.

FIG. 3A indicates a clear differentiation and sample grouping for thedifferent stages of parasitemia (i.e. R, T & G) from infected RBCscompared to the control (C) in the C—H stretching region. Ring stageparasites that are to the right of the Scores Plot have a large positivePC1 value compared to all other stages indicating significantdifferences in the lipid composition compared to the other stages. Thelinear sub-groupings observed in the clusters arise from the fact that aseries of concentrations were used as input data into the PCA. In thering stage four independent series were included in the PCA model.

FIG. 3B shows the Loadings Plot for PC1, which displays negativeloadings associated with vibrational modes of lipid in the regions of2888-2880, 3060-2950 cm⁻¹ as expected from previous findings (Webster etal, Analytical chemistry 2009, 81. 2516-2524.)

PCA analysis was also applied to the 1800-1000 cm⁻¹ region for allstages where the Hz bands are expected (˜1712, 1664, and 1209 cm⁻¹)(data not shown). However, only a good, rather than excellent separationof the trophozoites and gametocytes from the control was achieved.

The ring stage parasites could not be as definitively separated from theother groups when using this spectral window because the rings only havevery small amounts of Hz. The definitive separation in the PCA ScoresPlot along with the linearity observed in the sub-groupings because ofthe different percentages of parastemia demonstrates that the CHstretching region (3100-2800 cm⁻¹) is ideal for PLS prediction models.

ATR-FTIR Sensitivity Using PCA Analysis

In order to examine the sensitivity of the ATR-FTIR to differentiateparasitemia at very low levels, PCA analysis was applied to the secondderivative spectra at the lowest % parasitemia in the sample series forboth rings & trophozoites versus control (as 0%). FIG. 4 shows anexample of the PCA analysis at 0.00001% parasitemia for rings versuscontrol in the C—H stretching region (3100-2800 cm−1).

In FIG. 4 the Scores Plot indicates a good separation or grouping ofrings at 0.00001% parasitemia (the lowest concentration prepared in thering series) versus control. The Loadings Plot for PC1 shows strongnegative loadings in the lipid band regions of 2854, 2954-2944, 2993 and3063 cm⁻¹.

Similar analysis was also performed for the gametocyte and trophozoiteseries at 0.09 and 0.5% (the lowest concentration available)respectively versus control, which exhibited an excellent separationbetween the gametocytes, trophozoites and the controls. The resultsconfirm the ability of ATR-FTIR to detect parasitemia levels down to0.00001%. The same type of PCA analysis was performed in the Hz region(1800-900 cm⁻¹), however, no separation was observed indicating the Hzregion is ineffective for diagnosing low levels of parasitemia.

PLS Models

Partial least squares (PLS) regression is a statistical method thatdevelops a linear regression model by projecting the predicted variable(% parasitemia) and the observable variable (spectra) onto a newmultidimensional space.

For the ring stage parasitemia three PLS models were constructed forthree ranges of parasitemia (at the ring stage) from 3 independenttrials namely model 1 (10-30%), model 2 (0-5%) and model 3 (0-1%) withthe lowest detectable parasitemia at 0.00001%. The PLS model is based ona full cross-validation model where one sample is left out and then theparasitemia of that sample predicted. The corresponding Root Mean SquareError of Validation (RMSEV) and R-squared values for each model are:model 1 (2.50 and 0.94), model 2 (0.32 and 0.95) and model 3 (0.07 and0.95).

The spectra presented in FIG. 5 show an example set of calibrationstandards for the ring stage parasites that was used to build the PLSregression model. To generate the final models calibration data from 3independent trials were incorporated.

The regression plots for the calibration and validation sets are shownin FIG. 6 along with the corresponding regression co-efficient plots for8-factors. The maxima and minima bands in the regression co-efficientplots show the bands that are important in generating the linearity inthe model. These correspond to the major bands associated with the lipidCH stretching vibrations (FIG. 6). Thus, the linearity of the model isbased on real spectral changes and not spectral artifacts such asbaseline modulations or noise. It was found that the best predictionswere obtained when using the lipid CH stretching region (3100-2800 cm−1)as opposed to the 1800-900 cm−1 region, where the majority of bands arepresent.

Earlier studies have also reported unique lipids associated with themalaria parasite. Studies have shown that neutral lipids accumulate inthe digestive compartment and in neutral lipid bodies during parasitedevelopment (Jackson et al, Food vacuole-associated lipid bodies andheterogeneous lipid environments in the malaria parasite, Plasmodiumfalciparum. Molecular microbiology 2004, 54. 109-122; Pisciotta et al,The role of neutral lipid nanospheres in Plasmodium falciparum haemcrystallization. Biochem. J 2007, 402. 197-204; and Ambele & Egan,Neutral lipids associated with haemozoin mediate efficient and rapidβ-haematin formation at physiological pH, temperature and ioniccomposition. Malaria Journal 2012, 11. 337.)

Images of parasites at the ring stage show small Hz crystals surroundedby neutral lipid spheres inside the digestive vacuole compared to athinner rim of lipids that surrounds a much larger Hz crystal at thelater trophozoite stage. Jackson et al (Molecular microbiology 2004, 54.109-122) demonstrated that neutral lipid bodies contain di- andtriacylglycerols and hypothesized that these structures act as storagecompartments for lipid by-products formed by phospholipid digestion inthe parasite's digestive vacuole. The Hz aliphatic and aromatic CHvibrations also contribute to this lipid spectral region enhancing theoverall sensitivity of the technique.

Using the same method for each series of infected RBCs at differentstages and percentages of parasitemia, a range of optimum regressionmodels were obtained for both lipid and Hz band ranges with the minimumnumber of factors and highest model fitness. Results indicated thatspectral pre-processing and removal of outliers improved the correlationcoefficient between predicted and measured values at lower factors whichgives the optimized model with minimum error to be considered as the“best” theoretical fit. The results with second derivatives alsoindicated further improvement because lower factors were required toachieve high correlation coefficients.

In addition to the above final models from large datasets, a summary ofthe results from optimum PLS models at C-H stretching region on RBCs ofdifferent parasitemia series and at different stages of parasitic lifecycle are given in Table 2. It summarizes all the prediction modelsapplied to the ATR-FTIR spectra of ring, trophozoite and gametocytesseries as well as combinations of all the series from different stagesof parasitemia after the data pre-processing was applied to each model.

TABLE 2 Summary of optimum PLS models at C—H stretching region (3100-2800 cm⁻¹) on parasite (P) series: rings (R), trophozoites (T),gametocytes (G) & control (C) and combined series from R, T, G & C.Range of PLS-Conc. R-squared Range of RMSEP* P-Type & series Range (% P)Factor 1 to 7 Factor 1 to 7 R (d)    0-0.0001 0.54-0.32 0.00025-0.00027R (d)     0-0.00005 0.83-0.52  0.00008-0.000012 R (c)  0-0.1 0.993-0.3970.003-0.028 R (a, b) 0-5 0.99-0.21 0.16-1.55 R (c) 0-7 0.970-0.1530.1590.858 R (c)  5-10 0.991-0.151 0.175-1.73  R (a, c)  0-100.988-0.32  0.36-2.77 R (a)  5-30 0.997-0.56  0.46-6.9  R (a) 10-300.998-0.55  0.45-6.87 T 0-5 0.976-266  0.14-1.6  T  5-20 0.999-0.73 0.186-0.7  T 20-80 0.995-0.633  1.47-11.04 R (a), T, C  0-80  0.86-0.027 8.5-22.1 R (a), G, C 0-5 0.989-0.40  0.185-1.4  R (a), T, G, C 0-50.96-0.97 0.17-0.82 G 0-5  0.91-0.155  0.5-1.58 G 10-80 0.93-0.25 6.12-20.16 G 0-5 0.996-0.13  0.09-1.47 G 0-5 0.999-0.3   0.3-11.7 *RootMean Square Error of Predictions

Application of PLS Prediction Models to Unknown/Blind Samples

To assess the applicability and sensitivity of the PLS models, optimizedPLS prediction models from low ranges of parasitemia series (0-5, 0-0.1and 5-10%) were used to predict parasitemia concentration of a series ofinfected RBCs with rings as unknown or blind samples.

The replicate spectra (10-15) of the unknown samples were pre-processedin the same way as the reference samples and used for the PLSprediction. Both PLS models from rings series (with R2>0.99 andRMSE<0.17) as well as from combined spectra from all series were usedfor predictions. FIG. 7 indicates an example of predictions where ringsamples at 7 & 7.4% parasitemia were used as unknown and a PLS model inthe range of 5-10% parasitemia was used, the average standard error ofthe prediction-deviations was 0.08% at factor 1.

More ring samples with parasitemia levels in the range of 0 to 2 werealso used as unknowns. The average predicted concentrations of the ringsamples were all within 0-2% with average error of 0.2 (Hotelling T² at95% confidence limit). Predictions for the unknown samples with <0.1%parasitemia showed an average standard deviation of 0.05.

The reasons for the prediction variation especially at low parasitemialevels could be due to; (i) the varying number of infected cellsdeposited on the ATR diamond cell that may have caused non-uniformity inlipid distribution in the dried-sample deposits, (ii) varying thicknessof the lipid deposit at the crystal surface (iii) the error involved inthe sample preparation (e.g. separation and dilution) and the referencemethod.

Sample uniformity, particle size and consistency in sample thickness onthe diamond cell were found to be the most important factors inobtaining consistent spectral acquisition and error reduction forprediction of unknown samples. The detection limit or sensitivity of thepredictions was found to be 0.2% within 95% confidence limit.

The experimental results described thus demonstrate the utility of ATRFTIR spectroscopy as a method for rapid detection and quantification ofmalaria parasite infections.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodification(s). This application is intended to cover any variationsuses or adaptations of the invention following in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

As the present invention may be embodied in several forms withoutdeparting from the spirit of the essential characteristics of theinvention, it should be understood that the above described embodimentsare not to limit the present invention unless otherwise specified, butrather should be construed broadly within the spirit and scope of theinvention as defined in the appended claims. The described embodimentsare to be considered in all respects as illustrative only and notrestrictive.

Various modifications and equivalent arrangements are intended to beincluded within the spirit and scope of the invention and appendedclaims. Therefore, the specific embodiments are to be understood to beillustrative of the many ways in which the principles of the presentinvention may be practiced. In the following claims, means-plus-functionclauses are intended to cover structures as performing the definedfunction and not only structural equivalents, but also equivalentstructures.

It should be noted that where the terms “server”, “secure server” orsimilar terms are used herein, a communication device is described thatmay be used in a communication system, unless the context otherwiserequires, and should not be construed to limit the present invention toany particular communication device type. Thus, a communication devicemay include, without limitation, a bridge, router, bridge-router(router), switch, node, or other communication device, which may or maynot be secure.

Various embodiments of the invention may be embodied in many differentforms, including computer program logic for use with a processor (e.g.,a microprocessor, microcontroller, digital signal processor, or generalpurpose computer and for that matter, any commercial processor may beused to implement the embodiments of the invention either as a singleprocessor, serial or parallel set of processors in the system and, assuch, examples of commercial processors include, but are not limited toMerced™, Pentium™, Pentium II™ Xeon™, Celeron™, Pentium Pro™, Efficeon™,Athlon™, AMD™ and the like), programmable logic for use with aprogrammable logic device (e.g., a Field Programmable Gate Array (FPGA)or other PLD), discrete components, integrated circuitry (e.g., anApplication Specific Integrated Circuit (ASIC)), or any other meansincluding any combination thereof.

In an exemplary embodiment of the present invention, predominantly allof the communication between users and the server is implemented as aset of computer program instructions that is converted into a computerexecutable form, stored as such in a computer readable medium, andexecuted by a microprocessor under the control of an operating system.

Computer program logic implementing all or part of the functionalitywhere described herein may be embodied in various forms, including asource code form, a computer executable form, and various intermediateforms (e.g., forms generated by an assembler, compiler, linker, orlocator). Source code may include a series of computer programinstructions implemented in any of various programming languages (e.g.,an object code, an assembly language, or a high-level language such asFortran, C, C++, JAVA, or HTML. Moreover, there are hundreds ofavailable computer languages that may be used to implement embodimentsof the invention, among the more common being Ada; Algol; APL; awk;Basic; C; C++; Conol; Delphi; Eiffel; Euphoria; Forth; Fortran; HTML;Icon; Java; Javascript; Lisp; Logo; Mathematica; MatLab; Miranda;Modula-2; Oberon; Pascal; Perl; PL/I; Prolog; Python; Rexx; SAS; Scheme;sed; Simula; Smalltalk; Snobol; SQL; Visual Basic; Visual C++; Linux andXML.) for use with various operating systems or operating environments.The source code may define and use various data structures andcommunication messages. The source code may be in a computer executableform (e.g., via an interpreter), or the source code may be converted(e.g., via a translator, assembler, or compiler) into a computerexecutable form.

The computer program may be fixed in any form (e.g., source code form,computer executable form, or an intermediate form) either permanently ortransitorily in a tangible storage medium, such as a semiconductormemory device (e.g, a RAM, ROM, PROM, EEPROM, or Flash-ProgrammableRAM), a magnetic memory device (e.g., a diskette or fixed disk), anoptical memory device (e.g., a CD-ROM or DVD-ROM), a PC card (e.g.,PCMCIA card), or other memory device. The computer program may be fixedin any form in a signal that is transmittable to a computer using any ofvarious communication technologies, including, but in no way limited to,analog technologies, digital technologies, optical technologies,wireless technologies (e.g., Bluetooth), networking technologies, andinter-networking technologies. The computer program may be distributedin any form as a removable storage medium with accompanying printed orelectronic documentation (e.g., shrink wrapped software), preloaded witha computer system (e.g., on system ROM or fixed disk), or distributedfrom a server or electronic bulletin board over the communication system(e.g., the Internet or World Wide Web).

Hardware logic (including programmable logic for use with a programmablelogic device) implementing all or part of the functionality wheredescribed herein may be designed using traditional manual methods, ormay be designed, captured, simulated, or documented electronically usingvarious tools, such as Computer Aided Design (CAD), a hardwaredescription language (e.g., VHDL or AHDL), or a PLD programming language(e.g., PALASM, ABEL, or CUPL). Hardware logic may also be incorporatedinto display screens for implementing embodiments of the invention andwhich may be segmented display screens, analogue display screens,digital display screens, CRTs, LED screens, Plasma screens, liquidcrystal diode screen, and the like.

Programmable logic may be fixed either permanently or transitorily in atangible storage medium, such as a semiconductor memory device (e.g., aRAM, ROM, PROM, EEPROM, or Flash-Programmable RAM), a magnetic memorydevice (e.g., a diskette or fixed disk), an optical memory device (e.g.,a CD-ROM or DVD-ROM), or other memory device. The programmable logic maybe fixed in a signal that is transmittable to a computer using any ofvarious communication technologies, including, but in no way limited to,analog technologies, digital technologies, optical technologies,wireless technologies (e.g., Bluetooth), networking technologies, andinternetworking technologies. The programmable logic may be distributedas a removable storage medium with accompanying printed or electronicdocumentation (e.g., shrink wrapped software), preloaded with a computersystem (e.g., on system ROM or fixed disk), or distributed from a serveror electronic bulletin board over the communication system (e.g., theInternet or World Wide Web).

“Comprises/comprising” and “includes/including” when used in thisspecification is taken to specify the presence of stated features,integers, steps or components but does not preclude the presence oraddition of one or more other features, integers, steps, components orgroups thereof. Thus, unless the context clearly requires otherwise,throughout the description and the claims, the words ‘comprise’,‘comprising’, ‘includes’, ‘including’ and the like are to be construedin an inclusive sense as opposed to an exclusive or exhaustive sense;that is to say, in the sense of “including, but not limited to”.

1. A method of detecting parasitemia with an ATR-FTIR spectrometer comprising: (i) delivering an evanescent IR beam using an ATR substrate of an ATR-FTIR spectrometer in contact with a patient sample, wherein the patient sample is dried onto the ATR substrate; (ii) detecting IR radiation transmitted from the ATR substrate to produce a spectrum of the patient sample; (iii) processing one or more signals in the spectrum to identify any parasite; and (iv) determining a parasitemia level of the patient sample using one or more signals in the spectrum.
 2. The method according to claim 1 wherein the parasitemia level is ≥100 parasites/μl of sample.
 3. The method according to claim 1 wherein the parasitemia level is ≥50 parasites/μl of sample.
 4. The method of claim 1, further comprising transmitting the spectrum from a remote location to a server.
 5. The method of claim 4, wherein processing one or more signals in the spectrum is performed on the server.
 6. The method of claim 4, wherein determining the parasitemia level is performed on the server.
 7. The method of claim 1, wherein the processing comprises converting the spectrum into a second derivative, then applying a partial least squares regression model generated by using a library comprising a calibration set of spectral standards containing mixtures of normal and infected patient samples at different ratios.
 8. The method of claim 7, further comprising transmitting the spectrum from a remote location to a server, and wherein the processing is performed at the server.
 9. The method of claim 1, wherein the processing of the signal is performed via execution by the processor of a predetermined instruction set stored in a computer readable medium.
 10. The method of claim 1, further comprising fixing the sample in methanol.
 11. The method of claim 10, further comprising drying the sample using a heat source.
 12. The method of claim 1, wherein the one or more signals in the spectra are one more signals in the spectra at 2850, 2854, 2873, 2904, 2908, 2942, 2978, 2982, 2985, 3012 or 3040 cm−1.
 13. The method of claim 1, wherein the one or more signals in the spectrum comprise different spectra at different parasitemia levels.
 14. The method of claim 1, wherein the one or more signals in the spectrum comprise at least one signal at 2854, 2904, 2942 or 2982 cm−1 to quantify a parasitemia level between 10-30%.
 15. The method of claim 1, wherein the one or more signals in the spectrum comprise signals at 2854, 2904, 2942 and 2982 cm−1 to quantify a parasitemia level between 10-30%.
 16. The method of claim 1, wherein the one or more signals in the spectra comprise at least one signal of at 2873, 2908, 2978 or 3012 cm−1 to quantify a parasitemia level between 0-5%.
 17. The method of claim 1, wherein the one or more signals in the spectrum comprise signals at 2873, 2908, 2978 and 3012 cm−1 to quantify a parasitemia level between 0-5%.
 18. The method of claim 1, wherein the one or more signals in the spectrum comprise at least one signal of at 2857, 2942, 2985 or 3040 cm−1 to quantify a parasitemia level between 0-1%.
 19. The method of claim 1, wherein the one or more signals in the spectra comprise signals at 2857, 2942, 2985 and 3040 cm−1 to detect the presence of any parasitemia.
 20. A method, comprising: with a processor, executing an instruction set stored in a non-transitory computer readable storage medium, the instruction set including instructions to compare one or more spectral signals characteristic of an infected patient sample to detect matches and quantify a parasitemia level using different wavenumber values or absorbance values for different infection levels.
 21. The method of claim 20, wherein the one or more signals are spectral bands at 2850, 2854, 2873, 2904, 2908, 2942, 2978, 2982, 2985, 3012 or 3040 cm−1.
 22. The method of claim 20, wherein the one or more signals comprise different spectra at different parasitemia levels.
 23. A system of analysis for diagnosing infection, the system comprising: an ATR substrate for receiving a patient fluid sample; an FTIR spectrometer configured to generate an evanescent wave that extends into the sample using the ATR substrate; a detector for detecting IR radiation transmitted from the ATR substrate to produce a signal; and a processor configured to process the signal to create an FTIR spectrum of the patient sample and determine an infection level using different wavenumber values for different infection levels.
 24. The system of claim 23, wherein the processor is located in a server and the ATR substrate, FTIR spectrometer and detector are at a remote location from the processor. 